Team:Paris/DryLab

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== DryLab ==
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<center> [[team:Paris/DryLab#DryLab| Main]] - [[team:Paris/Modeling#bottom|Introduction]]
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- [[Team:Paris/Production_modeling2#bottom | Vesicle model]] - [[Team:Paris/Production_modeling#bottom | Delay model]]  - [[Team:Paris/Transduction_modeling#bottom | Fec simulation]] </center>
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== DryLab - Main==
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<center> '''Main'''</center>
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== Main ==
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<a class="menu_sub_active"href="https://2009.igem.org/Team:Paris/DryLab#bottom"> Main </a>|
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Production_modeling#bottom"> Delay model</a>|
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Production_modeling2#bottom"> Vesicle model</a>|
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<a class="menu_sub"href="https://2009.igem.org/Team:Paris/Transduction_modeling#bottom"> Fec simulation</a>
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===Questions===
===Questions===
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Our ''Message in a Bubble'' project aims at developing a communication system between bacterias. Among the various problems raised during the design phase, we investigated 3 of them with modeling and simulation:
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Our ''Message in a Bubble'' project aims at developing a communication system between bacteria. Among the various problems raised during the design phase, we investigated 3 of them with modeling and simulation:
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*'''What is the link between Tol/Pal expression and vesicule formation?''' Or, more precisely, '''can we explain vesicule formation solely by the diffusion of the doubly anchored Tol/Pal complex in the membrane ?'''. Understanding this connection is instrumental, since our project relies on the hypothesis that an increased in vesicule formation can be obtained simply by destabilization of the Tol/Pal complexes.  
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*'''What is the link between Tol/Pal expression and vesicle formation?''' Or, more precisely, '''can we explain vesicule formation solely by the diffusion of the doubly anchored Tol/Pal complex in the membrane ?''' Understanding this connection is instrumental, since our project relies on the hypothesis that an increased in vesicle formation can be obtained simply by destabilization of the Tol/Pal complexes.  
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*'''How to improve the quality of the signal sent?''' Or more precisely, '''how can we get a good synchronisation between the maximal production rate of vesicules and the maximal concentration of proteins to encapsulate ?'''
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*'''How to improve the quality of the signal sent?''' Or more precisely, '''how can we get a good synchronization between the maximal production rate of vesicles and the maximal concentration of proteins to encapsulate ?'''  
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*'''How to optimize the quality of the reception?''' Or more precisely, '''how can we get a robust response despite  a potentially very low number of signals (ie, vesicles) recieved ?'''
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*'''How to optimize the quality of the reception?''' Or more precisely, '''how can we get a robust response despite  a potentially very low number of signals (ie, vesicles) received ?''' Two characteristics of the response have been investigated: response amplitude and response time.
===Results===
===Results===
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*'''Understanding the link between Tol/Pal expression and vesicule formation'''
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To create a delay between the maximum concentration of proteins and of the maximum creation of vesicules by time units, we used a transcriptional cascade inside the genetic network thus synchronising protein and vesicule creations.
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Overall, we showed that the '''formation of vesicles can be simply explained by the diffusion of Tol/Pal protein complexes'''.
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This very surprising result comes from 3 specific phenomena:
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:* The initial formation of small ''blebs'' can be explained by ''differences of osmotic pressures'' between intra- and extra-cellular environments, together with a non-perfectly uniform distribution of Tol/Pal complexes acting like ''press studs''. (<u>[[Team:Paris/Production modeling2#top|Details]]</u> and figure 1)
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:* At the basis of nascent blebs, where the outer membrane is ''non-flat'', the simple diffusion (ie Brownian motion) of ''doubly-anchored'' Tol/Pal molecules leads to their accumulation. (<u>[[Team:Paris/Production modeling2#top|Details]]</u> and figure 2)
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:* Rings of accumulated Tol/Pal at the basis of nascent blebs tend to ''constrict'': '''vesiculation happens'''. This again simply results from plain Brownian motion on non-flat surfaces. (<u>[[Team:Paris/Production modeling2#top|Details]]</u>)
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[[Image:Delay System.jpg|800px|center]]
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[[Image:Vesicle_wiki.jpg|200px|center]]
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<center>
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Figure1: Initial formation of small ''blebs'' resulting from intra- and extra-cellular osmotic pressures differences combined with non-perfectly uniform distribution of Tol/Pal complexes acting like ''press studs'' between the internal membrane and the outer membrane.
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</center>
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[[Image:Accumulation3.png|600px|center]]
<center>
<center>
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Plot showing that vesiculation happens only once the protein encapsulated has reached its maximal concentration.
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Figure2: the simple diffusion of proteins on a non-flat surface leads to their accumulation at region of negative curvature (as it is the case at the basis of blebs).
</center>
</center>
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*'''Improving the quality of the signal sent'''
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Indeed, the over expression of TolRII takes an important part in the creation of vesicles, disturbing the Tol-Pal system which '''act as a physical anchor for the outer membrane to the cell-wall''':
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In order to obtain a good synchronization between the maximal production rate of vesicles and the maximal concentration of proteins to encapsulate, we have to introduce a '''delay''' between the initiation of the synthesis of the proteins to encapsulate and the initiation of the synthesis of the truncated Tol protein that causes vesicle formation.
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We showed that this delay can be conveniently implemented by using a  '''transcriptional cascade'''. More details  <u>[[Team:Paris/Production_modeling#bottom|here]]</u>
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:*We '''demonstrate''' (<u>[[Team:Paris/Production modeling2#top|Details]]</u>) that the formation of ''blebbing'' is due to this anchor system and to the osmotic pressure increase caused by the peptidoglycan turnover.
 
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[[Image:Vesicle_wiki.jpg|400px|center]]
 
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[[Image:Delay System.jpg|400px|center]]
<center>
<center>
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figure: Blebbing simulation obtain for random Tol/Pal distribution.
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Figure 4: Plot showing that vesiculation happens only when the protein to encapsulate has reached its maximal concentration.
</center>
</center>
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*'''Optimizing the quality of message reception'''
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Using stochastic simulations we identified two possible problems causing a non-robust response when the messenger concentration is low:
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* the activation does not always occur
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* even when it occurs, the activation time can vary significantly.
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:*We show that an accumulation of protein in the aera of negative curvature is explainable by simple diffusion mechanism.
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After trying various solutions, we found that an '''overexpression of the FecR protein''' solves both problems:
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simulations showed a good and robust activation in this case. More details <u>[[Team:Paris/Transduction_modeling#bottom|here]]</u>
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:*We show that this two phenomenon linked together can explains '''the whole maturation of a blebbing in a vesicle'''.
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Concerning reception, stochastic simulations revealed two possible problems reducing the robustness of our reception system :
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*when the amount of messengers received is too weak, the activation does not always occur
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*even when it occurs, the activation time can vary.
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[[Image:Stochstic inactivation.jpg|395px|left]]
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[[Image:Main01.png|395px|right]]
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<center>
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Figure 5: Plot showing that a more robust response is obtained when FecR is overexpressed in receptor cells.
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</center>
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After trying various solutions in our modeling study, we proposed to introduce an over expression of FecR protein to solve this problem ; simulations showed a good and robust activation in this case.
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{{Template:Paris2009_guided|Project#top|Production_modeling#bottom}}

Latest revision as of 03:55, 22 October 2009

iGEM > Paris > Home > DryLab > Main


DryLab - Main

Questions

Our Message in a Bubble project aims at developing a communication system between bacteria. Among the various problems raised during the design phase, we investigated 3 of them with modeling and simulation:

  • What is the link between Tol/Pal expression and vesicle formation? Or, more precisely, can we explain vesicule formation solely by the diffusion of the doubly anchored Tol/Pal complex in the membrane ? Understanding this connection is instrumental, since our project relies on the hypothesis that an increased in vesicle formation can be obtained simply by destabilization of the Tol/Pal complexes.


  • How to improve the quality of the signal sent? Or more precisely, how can we get a good synchronization between the maximal production rate of vesicles and the maximal concentration of proteins to encapsulate ?


  • How to optimize the quality of the reception? Or more precisely, how can we get a robust response despite a potentially very low number of signals (ie, vesicles) received ? Two characteristics of the response have been investigated: response amplitude and response time.

Results

  • Understanding the link between Tol/Pal expression and vesicule formation

Overall, we showed that the formation of vesicles can be simply explained by the diffusion of Tol/Pal protein complexes. This very surprising result comes from 3 specific phenomena:

  • The initial formation of small blebs can be explained by differences of osmotic pressures between intra- and extra-cellular environments, together with a non-perfectly uniform distribution of Tol/Pal complexes acting like press studs. (Details and figure 1)
  • At the basis of nascent blebs, where the outer membrane is non-flat, the simple diffusion (ie Brownian motion) of doubly-anchored Tol/Pal molecules leads to their accumulation. (Details and figure 2)
  • Rings of accumulated Tol/Pal at the basis of nascent blebs tend to constrict: vesiculation happens. This again simply results from plain Brownian motion on non-flat surfaces. (Details)


Vesicle wiki.jpg

Figure1: Initial formation of small blebs resulting from intra- and extra-cellular osmotic pressures differences combined with non-perfectly uniform distribution of Tol/Pal complexes acting like press studs between the internal membrane and the outer membrane.

Accumulation3.png

Figure2: the simple diffusion of proteins on a non-flat surface leads to their accumulation at region of negative curvature (as it is the case at the basis of blebs).


  • Improving the quality of the signal sent

In order to obtain a good synchronization between the maximal production rate of vesicles and the maximal concentration of proteins to encapsulate, we have to introduce a delay between the initiation of the synthesis of the proteins to encapsulate and the initiation of the synthesis of the truncated Tol protein that causes vesicle formation. We showed that this delay can be conveniently implemented by using a transcriptional cascade. More details here


Delay System.jpg

Figure 4: Plot showing that vesiculation happens only when the protein to encapsulate has reached its maximal concentration.

  • Optimizing the quality of message reception

Using stochastic simulations we identified two possible problems causing a non-robust response when the messenger concentration is low:

  • the activation does not always occur
  • even when it occurs, the activation time can vary significantly.

After trying various solutions, we found that an overexpression of the FecR protein solves both problems: simulations showed a good and robust activation in this case. More details here

Stochstic inactivation.jpg
Main01.png

Figure 5: Plot showing that a more robust response is obtained when FecR is overexpressed in receptor cells.


Open book.gif

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